Fig 2: Butyrate Promotes Antimicrobial Activity in Macrophages via HDAC Inhibitory Function(A–C) Gene expression of hcar2 (A), ffar2 (B), and ffar3 (C) on flow cytometry sorted CD19+ B, CD8+ T, naive CD4+ T, memory CD4+ T, CD56+ CD3- NK cells, CD14+ monocytes, CD141+ DCs, and CD1c+ DCs from the blood of healthy donors. Each dot represents one donor.(D) Gentamicin protection assay on control and butyrate macrophages, or butyrate macrophages differentiated in the presence of butyrate with pertussis toxin (PT).(E) Protein expression of acetyled histone 3 (Ac-H3) and acetyled histone 4 (Ac-H4) in control and butyrate macrophages (data from 2 individual donors per condition).(F and G) Intra-cellular expression of tri-methylated lysine27 on histone 3 (3MeH3K27) (F) and acetylated lysine 27 on histone 3 (AcH3K27) (G) protein on control and butyrate macrophages by flow cytometry.(H, J, L, and N) Gentamicin protection assay on control macrophages, butyrate macrophages, and macrophages differentiated in the presence of valproate, phenylbutyrate (H), SAHA (J), TMP195, and tubacin (L), SBHA, 1-naphthohydroxamic acid (NA), or RGFP966 (N).(I, K, M, and O) Gene expression of S100A8 in control macrophages, butyrate macrophages, and macrophages differentiated in the presence of valproate, phenylbutyrate (I), SAHA (K), TMP195, and tubacin (M), SBHA, 1-naphthohydroxamic acid (NA), or RGFP966 (O).(P) Gentamicin protection assay in control and butyrate macrophages with and without HDAC3 siRNA-mediated gene silencing.(Q) Extracellular acidification rate in control and butyrate macrophages, as well as in RGFP966 and TMP195 treated macrophages. Data represent the mean of three biological replicates.For the gentamicin protection assay, the percentage of CFU (of control) was calculated from mean value for control group.Each dot represents one independent donor. For pairwise comparison Mann-Whitney U test was performed and for multiple group comparisons a one-way ANOVA (Kruskal-Wallis test) was performed. *p < 0.05, **p < 0.01, and ***p < 0.001.Please also see Figure S5E.

Fig 3: Bulk PBMCs Return to Immune Quiescence by Visit 1, Related to Figure 1(A and B) Representative flow cytometry plots and frequencies of aß and ?d T cell subsets at Visit 1 (V1) in PBMCs from healthy control (HC) and SARS-CoV-2-recovered (CoV2+) individuals.(C and D) Representative flow cytometry plots and frequencies of CD4+ and CD8+ T cell effector/activation states (Ki67+, T-bet+, HLA-DR+CD38+) of total non-naive, memory CD45RA+CCR7+/- CD4+ or CD8+ T cells at V1 in HC and CoV2+ PBMCs.(E and F) Representative flow cytometry plots and frequencies of CD4+ memory and T-helper subsets at V1 in HC and CoV2+ PBMCs.(G and H) Representative flow cytometry plots and frequencies of cTfh (CXCR5+CD45RA-) and cTfh activation (ICOS+PD-1+) and helper (CXCR3+/-CCR6+/-) subsets at V1 in HC and CoV2+ PBMCs.(I) Frequency of B cells (CD19+CD3-) at V1 in HC and CoV2+ PBMCs.Statistics determined by two-tailed Mann-Whitney tests. Multiple testing correction significance cutoff at FDR = 0.05 is p value < 0.05. Error bars represent mean and SD. Data from two experiments.

Fig 4: NK Cell Gating Strategy and NK Cell Frequency, Related to Figure 2(A) Representative example gated on live CD14-CD19-CD3-CD4- lymphocytes; CD56 and CD16 are used to identify NK cells, discriminating between populations on the basis of CD56bright, dim and negative expression levels.(B) Boxplots of the percentage of NK cells out of live lymphocytes in individuals in the bnAb (red; n = 22), control (blue; n = 19) and HIV-seronegative (black; n = 22) groups. Each symbol represents data from an individual subject and the box-and-whisker plots show the median, quartiles and range.(C) ViSNE map of NK and T cell distribution based on CD3, CD4, CD8, CD56 and CD16 expression from compiled subjects in the bnAb, control and HIV seronegative group. CD4CD3+ T cells are shown in blue, CD8CD3+ T cells in orange, CD56+CD3- NK cells in red and the CD56-CD16+ NK cell subset in green.

Fig 5: NK Cells Reduce Tfh Numbers and Help for B Cells in an In Vitro Tfh-B Cell Co-culture SystemTotal NK cells and resting CD127hiCD25lowCXCR5+CD4+Tfh cells isolated from the peripheral blood of HIV seronegative donors (n = 6) were activated with IL-2/12/15/18 (NK cells) or SEB (Tfh) then mixed and co-cultured with autologous naive B cells at a B:Tfh:NK ratio of 2:1:5 in the presence of SEB.(A) Representative example of live Tfh cells (gated within CD3+CD4+CD19-CD56- cells), class-switched B cells and plasmablasts (both gated within live CD3-CD4-CD19+IgM-IgD- cells) in the presence or absence of NK cells measured 6 days later.(B and C) CD4 Tfh numbers (B) and division index (C).(D and E) Numbers of class-switched memory B cells (D) and plasmablasts (E).(F and G) Supernatant levels of total IgM (F) IgG (G). Paired data from Tfh-B cell co-cultures carried out in the presence and absence of NK cells with samples from individual donors are shown. *p < 0.05. Wilcoxon matched-pairs signed rank test; p values corrected for multiple comparison analysis.See also Figure S6.